Cushing's disease is caused by the pituitary hypersecretion of ACTH and occurs predominately in women. Patients are cured by surgical removal of an ACTH-producing adenoma, suggesting evolution and expansion of a genetically aberrant cell. However, hypothalamic dysfunction and excessive stimulation of anterior pituitary corticotrophs by one or more neurotransmitter substances may also lead to the development of corticortropic adenomas. To study the clonal composition of ACTH-producing pituitary adenomas, we used restriction fragment length polymorphism (RFLP) of two X-chromosome linked genes, hypoxanthine phosphoribosyl transferase (HPRT) and phosphoglycerate kinase (PGK), in conjunction with their methylation patterns. Tumors from female patients only were used because in all female cells only one X-chromosome remains active which can e detected by the methylation pattern. Out of 27 tumors analyzed, nine were heterozygous at the HPRT or PGK loci. The clonal analysis demonstrated a monoclonal pattern in six of these tumors, whereas a polyclonal pattern was observed in three tumors including a pituitary adenoma from a patient with the Nelson's syndrome. There was no correlation between the clonal composition and the clinical features of the tumors. Studies are also in progress to search for possible mutations in the members of the ras gene family. Specific regions of the three ras genes, H-, K-, and N-ras, including the mutational hot spots in codons 12, 13, and 61 were amplified by the polymerase chain reaction chain reaction, PCR, and will be directly sequenced using the dideoxynucleotide chain termination method.